Relaxation Optimized Heteronuclear Experiments for Extending the Size Limit of RNA Nuclear Magnetic Resonance

The application of NMR to large RNAs has been limited by the inability to perform heteronuclear correlation experiments essential for resolving overlapping ¹H NMR signals, determining interproton distance restraints and interhelical orientations for structure calculations, and evaluating conformational dynamics. Approaches exploiting ¹H–¹³C correlations that are routinely applied to proteins and small RNAs of ∼60 nucleotides or fewer are impractical for larger RNAs due to rapid dipolar relaxation of protons by their attached carbons. Here we report a ²H-enhanced, ¹H–¹⁵N correlation approach that enables atom-specific NMR characterization of much larger RNAs. Purine H8 transverse relaxation rates are reduced ∼20-fold with ribose perdeuteration, enabling efficient magnetization transfer via two-bond ¹H–¹⁵N couplings. We focus on H8–N9 correlation spectra which benefit from favorable N9 chemical shift anisotropy. Chemical shift assignment is enabled by retention of protons at the C1′ position, which allow measurement of two-bond H1′–N9 and through-space H1′–H8 correlations with only a minor effect on H8 relaxation. The approach is demonstrated for the 232 nucleotide HIV-1 Rev response element, where chemical shift assignments, ¹⁵N-edited nuclear Overhauser effects, and ¹H–¹⁵N residual dipolar couplings are readily obtained from sensitive, high-resolution spectra. Heteronuclear correlated NMR methods that have been essential for the study of proteins can now be extended to RNAs of at least 78 kDa.